The present invention relates generally to reverse-turn mimetics, including inhibitors of cell adhesion-mediated disease, as well as to a chemical library of reverse-turn mimetics.
Cell adhesion is critical to the viability of living organisms. Adhesion holds multicellular tissues together and directs embryonic development. It plays important roles in wound healing, eradication of infection and blood coagulation. Integrins are a family of cell surface proteins intimately involved in all of these functions. They have been found in nearly every type of human cell except red blood cells. Abnormalities in integrin function contribute to a variety of disorders including inflammatory diseases, heart attack, stroke, and cancer.
Integrins consist of heterodimers of xcex1 and xcex2 subunits, non-covalently bound to each other. These cell surface receptors extend through the cell membrane into the cytoplasm. At least 15 different xcex1 and 9 different xcex2 subunits are known. However, because most xcex1 proteins associate with only a single xcex2 there are about 21 known integrin receptors. On the cell surface the heads of the two subunits contact each other to form a binding surface for extracellular protein ligands, allowing attachment to other cells or to the extracellular matrix. The affinity of these receptors may be regulated by signals from outside or within the cell. For example, recruitment of leukocytes to the site of injury or infection involves a series of adhesive interactions. Weak interaction between endothelial and leukocyte selectins and carbohydrates mediate transient adhesion and rolling of the leukocyte along the vessel wall. Various chemokines and other trigger factors released by the site of inflammation serve as signals to activate integrins from a quiescent to a high affinity state. These activated integrins then bind their cognate ligands on the surface of the endothelial cells, resulting in strong adhesion and flattening of the leukocyte. Subsequently the leukocyte migrates through the endothelium into the tissue below.
Integrin xcex14xcex21 mediates cell adhesion primarily through binding to either vascular cell adhesion molecule-1 (VCAM-1) or an alternatively spliced variant of fibronectin containing the type III connecting segment (IIICS). A variety of cells involved in inflammation express xcex14xcex21, including lymphocytes, monocytes, basophils and eosinophils, but not neutrophils. Monoclonal antibodies to the xcex14 subunit have been used to validate xcex14-containing integrins as potential therapeutic targets in animal models of rheumatoid arthritis (Barbadillo et al. Springer Semin Immunopathol 16: 427-36, 1995; Issekutz et al. Immunology 88: 569-76, 1996), acute colitis (Podolsky et al. J Clin Invest 92: 372-80, 1993), multiple sclerosis (Yednock et al. Nature 356: 63-6, 1992), asthma (Abraham et al. J. Clin. Invest. 93: 776-87, 1994; U.S. Pat. No. 5,871,734) and diabetes (Tsukamoto et al. Cell Immunol 165: 193-201, 1995). More recently, low molecular weight peptidyl derivatives have been produced as competitive inhibitors of xcex14xcex21 and one has been shown to inhibit allergic airway responses in sheep (Lin et al. J Med Chem 42: 920-34, 1999).
It has been shown that a key sequence in IIICS involved in binding to xcex14xcex21 is the 25 residue peptide CS1, and within that sequence the minimally recognized motif is the tripeptide, LDV. A similar sequence, IDS, has been implicated in the binding of VCAM-1 to xcex14xcex21. X-ray crystal structures of an N-terminal two-domain fragment of VCAM-1 show that the IDS sequence is part of an exposed loop linking two beta-strands (Jones et al. Nature 373: 539-44, 1995; Wang et al. Proc Natl Acad Sci U S A 92: 5714-8, 1995). Cyclic peptides and derivatives thereof which adopt reverse-turn conformations have proven to be inhibitors of VCAM-1 binding to xcex14xcex21 (WO 96/00581; WO 96/06108; Doyle et al. Int J Pept Protein Res 47: 427-36, 1996). In addition, a number of potent and selective (versus xcex15xcex21) cyclic peptide-based inhibitors have been discovered (Jackson et al. J Med Chem 40: 3359-68, 1997). Several non-peptidyl beta-turn mimetics have also been reported to bind xcex14xcex21 with IC50s in the low micromolar range (Souers et al. Bioorg Med Chem Lett 8: 2297-302, 1998). Numerous phenylalanine and tyrosine derivatives have also been disclosed as inhibitors of xcex14xcex21 (WO 99/06390; WO 99/06431; WO 99/06433; WO 99/06434; WO 99/06435; WO 99/06436; WO 99/06437; WO 98/54207; WO 99/10312; WO 99/10313; WO 98/53814; WO 98/53817; WO 98/58902) However, no potent and orally available small molecule inhibitors have been disclosed.
A related integrin, xcex14xcex27, is expressed on the surface of lymphocytes and binds VCAM-1, fibronectin and mucosal addressin cell adhesion molecule 1 (MAdCAM-1). Integrin xcex14xcex27 and MAdCAM mediate recirculation of a subset of lymphocytes between the blood, gut, and lymphoid tissue. Similar to VCAM-1 and Fibronectin CS-1 there is a tripeptide sequence, LDT, present on the CD loop of MAdCAM-1 which is important for recognition by xcex14xcex27. An X-ray crystal structure shows this sequence is also part of a turn structure (Tan et al. Structure 6: 793-801, 1998). Recent studies have shown that xcex14xcex27 may also play a part in diseases such as asthma (Lobb et al. Ann N Y Acad Sci 796: 113-23, 1996), inflammatory bowel disease (Fong et al. Immunol Res 16: 299-311, 1997), and diabetes (Yang et al. Diabetes 46: 1542-7, 1997). In addition, while xcex14 integrins appear to be down-regulated in carcinomas such as cervical and prostate, they appear to be up-regulated in metastatic melanoma (Sanders et al. Cancer Invest 16: 329-44, 1998), suggesting that inhibitors of xcex14xcex21 and xcex14xcex27 may be useful as anticancer agents.
Reverse-turns comprise one of three classes of protein secondary structure and display three (gamma-turn), four (beta-turns), or more (loops) amino acid side chains in a fixed spatial relationship to each other. Turns have proven important in molecular recognition events (Rose et al. Advances in Protein Chemistry 37: 1-109, 1985) and have engendered a burgeoning field of research into small molecule mimetics of them (e.g. Hanessian et al. Tetrahedron 53: 12789-12854, 1997). Many mimetics have either been external turn-mimetics which do not allow for the display of all the physiologically relevant side-chains (e.g. Freidinger et al. Science 210: 656-8, 1980) or small, conformationally mobile cyclic peptide derivatives (e.g. Viles et al. Eur J Biochem 242: 352-62, 1996). However, non-peptide compounds have been developed which closely mimic the secondary structure of reverse-turns found in biologically active proteins or peptides. For example, U.S. Pat. Nos. 5,475,085, 5,670,155 and 5,672,681, to Kahn and published PCT WO94/03494 to Kahn all disclose conformationally constrained, non-peptidic compounds which mimic the three-dimensional structure of reverse-turns. More recently, published PCT WO97/15577 to Kahn and PCT WO98/49168 to Kahn et al. have disclosed additional, highly constrained bicyclic heterocycles as reverse-turn mimetics. Nevertheless, as no one template can mimic every type of turn, there remains a need in the art for additional reverse-turn templates and methods for their use.
While significant advances have been made in the synthesis and identification of conformationally constrained, reverse-turn mimetics, there is still a need in the art for small molecules that mimic the secondary structure of peptides. In addition, there is a need in the art for techniques for synthesizing libraries of such mimetics and screening the library members against biological targets to identify bloactive library members. Further, there is a need in the art for small, orally available inhibitors of integrins, for use in treating inflammatory diseases and cardiovascular diseases, as well as some cancers. In particular there is a need for inhibitors of xcex14xcex21 and xcex14xcex27, for use in the treatment of rheumatoid arthritis, asthma, diabetes and inflammatory bowel disease. The present invention fulfills these needs, and provides further related advantages.
In brief, the present invention is directed to conformationally constrained compounds which mimic the secondary structure of reverse-turn regions of biologically active peptides and proteins. This invention also discloses libraries containing such compounds, as well as the synthesis and screening thereof. Furthermore, the invention discloses the use of reverse-turn mimetics for the treatment of cell adhesion-mediated diseases.
The compounds of the present invention have the following general structure (I): 
wherein Y is selected from xe2x80x94CH(R5)-A-N(R1)xe2x80x94, -A-N(R1)xe2x80x94CH(Rxe2x80x2)xe2x80x94, -A-N(R1)xe2x80x94C(xe2x95x90O)xe2x80x94, -A-C(xe2x95x90O)xe2x80x94N(R1)xe2x80x94, -A-CH(R1)xe2x80x94Oxe2x80x94, and -A-CH(R1)xe2x80x94N(Rxe2x80x2)xe2x80x94; A is xe2x80x94(CHRxe2x80x2)nxe2x80x94; B is xe2x80x94(CHRxe2x80x3)mxe2x80x94; n=0, 1 or 2; m=1, 2 or 3; and any two adjacent CH groups or adjacent NH and CH groups on the bicyclic ring may optionally form a double bond; and wherein Rxe2x80x2, Rxe2x80x3, R1, R2, R3, R4 and R5 are as defined in the following detailed description.
In the embodiment wherein Y is xe2x80x94CH(R5)-A-N(R1)xe2x80x94, the compounds of this invention have the following structure (Ixe2x80x2): 
wherein A and B are as defined above, and R1, R2, R3, R4 and R5 are as defined in the following detailed description.
In the embodiment wherein Y is -A-N(R1)xe2x80x94CH(Rxe2x80x2)xe2x80x94, the compounds of this invention have the following structure (Ixe2x80x3): 
wherein A and B are as defined above, and Rxe2x80x2, R1, R2, R3 and R4 are as defined in the following detailed description.
In the embodiment wherein Y is -A-N(R1)xe2x80x94C(xe2x95x90O)xe2x80x94, the compounds of this invention have the following structure (Ixe2x80x3xe2x80x2): 
wherein A and B are as defined above, and R1, R2, R3 and R4 are as defined in the following detailed description.
In the embodiment wherein Y is -A-C(xe2x95x90O)xe2x80x94N(R1)xe2x80x94, the compounds of this invention have the following structure (Ixe2x80x3xe2x80x3): 
wherein A and B are as defined above, and R1, R2, R3 and R4 are as defined in the following detailed description.
In the embodiment wherein Y is -A-CH(R1)xe2x80x94Oxe2x80x94, the compounds of this invention have the following structure (Ixe2x80x3xe2x80x3xe2x80x2): 
wherein A and B are as defined above, and R1, R2, R3 and R4 are as defined in the following detailed description.
In the embodiment wherein Y is -A-CH(R1)xe2x80x94N(Rxe2x80x2)xe2x80x94, the compounds of this invention have the following structure (Ixe2x80x3xe2x80x3xe2x80x3): 
wherein A and B are as defined above, and Rxe2x80x2, R1, R2, R3 and R4 are as defined in the following detailed description.
The present invention is also directed to libraries containing compounds of structure (I) above, as well as methods for synthesizing such libraries and methods for screening the same to identify biologically active compounds. Methods of use for treating cell-adhesion-mediated disease with the compounds of this invention are described. Compositions containing a compound of this invention in combination with a pharmaceutically acceptable carrier or diluent are also disclosed.
These and other aspects of this invention will be apparent upon reference to the attached figures and following detailed description. To this end, various references are set forth herein which describe in more detail certain procedures, compounds and/or compositions, and are incorporated by reference in their entirety.